Regenerative medicine, mainly utilizing stem cells, has considerably progressed in recent years. Various tissue stem cells, which had not been considered to be present, were discovered and identified in various tissues. Thus, attention has been focused on disease therapy using regenerative therapy.
However, regenerative therapy has not yet reached a point where it is conventionally applied to numerous patients suffering from organ or tissue dysfunction. To date, a very limited number of such patients have been treated by organ transplantation or use of an auxiliary medical system or apparatus. These therapies are problematic in terms of shortage of donors, rejection, infection, durability, and the like. In particular, the donor shortage raises serious problems. In the case of bone marrow transplantation, bone marrow and umbilical cord blood banks have gradually become more widely used at home and abroad, though it is still difficult to provide a limited amount of samples to the number of patients in need. Therefore, there is an increasing demand for therapies using stem cells and regenerative medicine using the same in order to overcome the above-described problems. Use of foreign tissue for organ implantation (e.g., heart, blood vessels, etc.) is hindered mainly by immune rejection responses. Changes occurring in allogeneic grafts (or allografts) and xenografts are well known.
After gastrulation, a fertilized egg is divided into three germ layers, i.e., endoderm, mesoderm, and ectoderm. Cells derived from the ectoderm are mainly present in brain, including neural stem cells and the like. Cells derived from the mesoderm are mainly present in bone marrow, including blood vessel stem cells, hematopoietic stem cells, mesenchymal stem cells, and the like. Cells derived from the endoderm are mainly present in organs, including liver stem cells, pancreatic stem cells, and the like.
Mesenchymal cells, such as adipocytes, bone cells, ligament cells, cardiac muscle cells, and the like, have an important function of forming the shape or skeleton of the body. Therefore, there is an increasing expectation for the application of groups of such cells or tissues of such cells to regenerative medicine and implantation medicine. Particularly, it has been reported that bone marrow mesenchymal stem cells can be differentiated into mesodermal organs, and such stem cells have attracted attention mainly in the field of regenerative medicine. However, differentiation of such cells requires special conditions where a special medium containing a differentiation inducing agent (e.g., dexamethasone, etc) is required (Nakatsuji, ed., “Kansaibo•Kuron Kenkyu Purotokoru [Stem cell/Clone Research Protocol]”, Yodosha (2001)).
Mesenchymal stem cells are a type of tissue stem cells. Mesenchymal stem cells naturally occur only in a small amount (one ten thousandth of all cells in the bone marrow of human neonates, thereafter reducing quickly, and one two millionth of all cells in elderly persons). It is therefore difficult to isolate mesenchymal stem cells. As it has been reported that mesenchymal stem cells are differentiated into germ layers other than mesoderm, the range of applications is becoming widespread. However, conditions for such differentiation are more specific than those which are described above. The known surface antigens of mesenchymal stem cells are CD105(+), CD73(+), CD29(+), CD44(+), CD14(−), CD34(−), and CD45(−).
Isolation of mesenchymal stem cells requires large costs. Further, taking bone marrow cells is generally associated with pain to the donor. Further, it is difficult to culture such cells without inducing differentiation serum from a specifically selected lot must be used thereby adding additional cost and labor to the use of such stem cells.
On the other hand, it has been found that fat contains stem cells (WO00/53795; WO03/022988; WO01/62901; Zuk, P. A., et al., Tissue Engineering, Vol. 7, 211-228, 2001; Zuk, P. A., et al., Molecular Biology of the Cell, Vol. 13, 4279-4295, 2002). A large amount of stem cells can be obtained from fat than other tissues (e.g., bone marrow, etc.) and the density of stem cells seem to be higher. Therefore, fat has attracted attention. Prior art methods for preparing stem cells from fat tissue (Japanese PCT National Phase Laid-Open Publication No. 2003-523267 and Japanese PCT National Phase Laid-Open Publication No. 2002-537849) have an advantage in availability in richer amount than when bone marrow is used as the cell source. However, it is necessary to treat the fat tissue, the cell source, with an enzyme such as collagenase, and therefore it is impossible to prepare stem cells in a simple and large amount. In considering application of stem cells and precursor cells to regenerative medicine, there is still demand for a method for preparing homogeneous stem cells and precursors in a simple manner and in a large amount.